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Prospective, non-interventional study carried out after culturing placental trophoblastic tissue ex vivo and infection with Zika and Tonate
The analysis will relate for each virus studied + control + favipiravir to 1 placenta in the 1st trimester and 1 placenta at term.
2 experiments per virus will be planned.
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| Label | Type | Description | Intervention Names |
|---|---|---|---|
| Human placentas and trophoblasts (biological waste) from uncomplicated pregnancies. | The placentas will be collected following a birth by caesarean section after oral and written information by delivery of the information note for women who have had a normal singleton pregnancy at term. Similarly, trophoblasts are collected following endo-uterine aspiration in the context of voluntary termination of pregnancy.
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| Name | Type | Description | Arm Group Labels | Other Names |
|---|---|---|---|---|
| Cell line and virus | Biological | C6/36 cells will be cultured in L15 culture medium (Leibovitz's L-15 medium), supplemented with 5% FBS (Fetal Bovine Serum) and 1% penicillin/streptomycin at 28°C in a humidified atmosphere containing 5% C02. ZIKV and TONV will be expanded and titrated using C6/36 cells. The viral stock will be aliquoted in 100 µL aliquots and stored at -80°C. |
| Measure | Description | Time Frame |
|---|---|---|
| To assess the replication potential of TONV and ZIKV after placenta infection ex vivo. | Determine the viral load (RT-qtPCR) of TONV and ZIKV after placenta infection, ex vivo in RNA copies/mL | inclusion |
| Measure | Description | Time Frame |
|---|---|---|
| Validate the ex vivo placental model for TONV. | Analysis of viral load in placental explants for TONV in RNA copies/mL | Inclusion |
| Compare viral infection of the placenta at different terms of pregnancy (first trimester and term). |
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Inclusion Criteria:
Exclusion Criteria:
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Pregnant women
| Name | Role | Phone | Extension | |
|---|---|---|---|---|
| PICONE Olivier | Contact | 01 47 60 66 36 | olivier.picone@aphp.fr | |
| DECHANET Aline | Contact | aline.dechanet@aphp.fr |
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| Infection of human placental culture explants | Biological | Placental tissues will be handled within one hour of delivery. The chorionic villi will be dissected into 5mm sections and the tissues washed abundantly, minimum 3 times with a standard culture medium (RPMI-1640 supplemented with 10% heat-inactivated fetal calf serum (FCS), 1% L-Glutamine and 1% Penicillin/Streptomycin) to remove maternal blood, membranes and blood clots. Collagen gel sponges will be placed in 6-well plate wells containing 3mL of culture medium (RPMI-1640 supplemented with 15% heat-inactivated fetal bovine serum (FCS), 1% Penicillin/streptomycin, 0 1% Gentamycin, 1% Amphotericin B, 1% L-Glutamine, 1% non-essential amino acid, 1% sodium pyruvate) per well. Chorionic villi will be dissected into 5mm sections and placed on top of collagen gel sponges at the interface between culture medium and air. |
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| Determination of viral load in tissues by qRT-PCR | Biological | The tissues will be lysed by mechanical disruption in a lysis buffer + 4%TCEP (Tris(2-carboxyethyl)phosphine hydrochloride) (Machery-Nagel ref: 740395.107) with the Precellys system. The lysed tissues will be diluted in 100 mg/mL of Macherey-Nagel lysis buffer, aliquoted and stored at -80°C until extraction. Total RNA will be extracted in duplicate using the Nucleospin 96 RNA Core kit (Macherey Nagel ref: 740466.4), following the supplier's instructions. Standard samples, controls and ZIKV and TONV viral RNA will be extracted and tested in parallel under the same conditions. The extracted RNA will be subjected to reverse transcriptase, using the Superscript One-Step RT-qPCR kit (Invitrogen ref: 11732088) according to the manufacturer's recommendations, with a probe and primer specific for ZIKV and TONV. |
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Analysis of the viral load according to the term in RNA copies/mL
| first trimester |
| Compare viral infection of the placenta at different terms of pregnancy (first trimester and term). | Analysis of gene expression of the different cytokines according to the term in RNA copies/mL | first trimester |
| Compare viral infection of the placenta at different terms of pregnancy (first trimester and term). | Analysis of the histological lesions according to the term in RNA copies/mL | first trimester |
| Compare viral infection of the placenta at different terms of pregnancy (first trimester and term). | Analysis of the viral load according to the term in RNA copies/mL | term |
| Compare the placental infection of TONV versus ZIKV. | Analysis of viral load depending on the virus (ZIKV vs TONV) in RNA copies/mL | inclusion |
| Compare the placental infection of TONV versus ZIKV. | Analysis of gene expression of different cytokines depending on the virus (ZIKV vs TONV) in RNA copies/mL | inclusion |
| Compare the placental infection of TONV versus ZIKV. | Analysis of histological lesions depending on the virus (ZIKV vs TONV) in RNA copies/mL | inclusion |
| Highlight the cellular targets of TONV. | Histological analysis and labeling of TONV cells in RNA copies/mL | inclusion |
| Highlight the cellular targets of TONV. | Histological analysis and labeling of placental cells in RNA copies/mL | inclusion |
| ID | Term |
|---|---|
| D000071243 | Zika Virus Infection |
| ID | Term |
|---|---|
| D000096724 | Mosquito-Borne Diseases |
| D000079426 | Vector Borne Diseases |
| D007239 | Infections |
| D001102 | Arbovirus Infections |
| D014777 | Virus Diseases |
| D018177 | Flavivirus Infections |
| D018178 | Flaviviridae Infections |
| D012327 | RNA Virus Infections |
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